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Abstract: FR-PO370

Drosophila Nephrocytes as a Personalized Platform for Validation of Variants in TBC1D8B-Associated Focal Segmental Glomerulosclerosis

Session Information

Category: Genetic Diseases of the Kidneys

  • 1102 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Milosavljevic, Julian, Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
  • Lempicki, Camille, Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
  • Lang, Konrad, Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
  • Heinkele, Helena, Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
  • Wang, Minxian, Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Knob, Andrea, Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Pollak, Martin, Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Hermle, Tobias F., Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
Background

Mutations in TBC1D8B have been discovered as a monogenic cause of nephrotic syndrome and functional studies in Drosophila support a role in nephrin trafficking. Nevertheless, the role of TBC1D8B remains incompletely understood. Frequency of TBC1D8B mutations among patients with hereditary FSGS is unclear while functional characterization of novel genetic variants continues to be challenging.

Methods

We tested evolutionary conservation of TBC1D8B through expression of murine Tbc1d8b in the background of a stable null allele of fly Tbc1d8b. We exploited the accessibility of slit diaphragms in podocyte-like Drosophila nephrocytes to assess Nephrin turnover in a Tbc1d8b loss-of-function. Novel TBC1D8B mutations were detected by whole exome sequencing within an FSGS cohort. Transgenesis of mammalian Tbc1d8b harboring patient mutations was used for validation in Drosophila in vivo.

Results

Fly Tbc1d8b was required for rapid nephrin turnover in nephrocytes and nephrin endocytosis induced by excessive function of Rab5. Accumulation of LAMP-GFP upon Tbc1d8b loss-of-function confirmed a role for endolysosomal degradation. Low level expression of murine Tbc1d8b rescued loss of the Drosophila gene demonstrating evolutionary conservation. We previously showed that excessive overexpression induces nephrin vesicles. To examine if this effect is linked to TBC/GAP function, we generated a transgene harboring a R537K mutation which targets the catalytic arginine finger. Expression of this transgene still entailed nephrin vesicles, but these were of a reduced size. This suggests that TBC1D8B function is partially GAP-independent. Finally, we discovered four novel TBC1D8B mutations within a cohort of 363 FSGS patients and validated a functional impact for three of these mutations in Drosophila studying nephrin vesicles in podocyte like nephrocytes.

Conclusion

The podocyte-like nephrocytes of Drosophila melanogaster are suitable for validation of novel variants and further functional characterization of the nephrotic syndrome gene TBC1D8B, establishing a personalized platform for TBC1D8B-associated FSGS.

Funding

  • Other NIH Support